1. Field of the Invention
The present invention relates generally to a method and apparatus for detecting biological activity. More particularly, the present invention relates to a method for making rapid analysis of materials in which the presence of microorganisms or the like is suspected by infrared analysis of a sample of the head space gas of a container in a sample cell located externally from the container.
When, for example, bacteria are cultured in a suitable medium including a carbon source, such as glucose, the carbon source is broken down to form CO.sub.2 during the growth and metabolism of the bacteria. It would be desirable to provide a rapid, sensitive method for the analysis of the gaseous atmosphere produced over the growth medium in the head space in order to determine the presence or absence of biological activity.
2. Description of the Prior Art
In many fields of endeavor it is important to be able to determine whether or not substances are contaminated with biologically active agents such as bacteria and the like. Examples of such fields are the medical field, the food processing industry, the pharmaceutical industry, the cosmetics industry and the field of public health
It has long been a standard practice to place a sample of material to be tested for the presence of biologically active agents on a semisolid nutrient medium contained in a Petri dish and to make visual observations of the resulting microbial growth, if any. A similar procedure involves the inoculation of a sterile vial or bottle of liquid nutrient medium with the suspect material, again followed by visual detection of growth. Not only are such methods slow and laborious, but because they depend upon the subjective judgment of individual human observers, the result obtained is not uniformly reliable.
Techniques have also been developed for the detection of bacteria which involve the incubation of a sample of material to be tested in a closed container with a radioactive isotope labeled culture medium with subsequent monitoring of the atmosphere in the container above the medium to determine whether or not radioactive gases are produced. A system of this type is disclosed in U.S. Pat. Nos. 3,676,679 and 3,935,073. Such systems are rapid and reliable, but they suffer from a number of disadvantages resulting primarily from the use of radioactive materials. Radioisotope labeled materials are expensive and require special handling during storage, use and disposal. Moreover, although the levels of radioactivity encountered in using such systems are very low, prospective users may be deterred by personal fears of radioactivity.
Systems have been described which do not require the use of radioactivity in any manner. U.S. Pat. No. 4,182,656 describes a method for the detection of biologically active agents based upon utilization of substrates enriched with stable Carbon-13. Although this method eliminates any requirement for radioisotopes in the detection system, nutrients enriched with Carbon-13 are less available and considerably more expensive than their radiolabled counterparts. Because .sup.13 CO.sub.2 has molecular properties very nearly the same as .sup.12 CO.sub.2, the most prevalent isotopic form of carbon dioxide, and because .sup.13 C comprises better than 1% of all carbon in the environment, special care must be taken to insure that .sup.13 C carbon dioxide is detected preferentially while ambient carbon dioxide is ignored. A mass spectrometer is generally used to detect changes in the relative abundance of stable isotopes, and was used in the development of the forementioned patent. Mass spectrometry requires relatively sophisticated high-vacuum instrumentation, and is thus not a suitable detection means for application in the typical microbiology laboratory.
U.S. Pat. No. 4,073,691 discloses a non-radiometric means for detection of biologically active agents through detection of any change in the character of the gas present over a liquid growth medium contained in a sealed vial system. Changes in the character of the gas are determined by measurement of the ratio of the selected product gas to an inert reference gas also present in the vial measurements made before and after the vial has been subjected to conditions conducive to bacterial growth. The inclusion of an inert reference gas for ratio measurement purposes requires that the detection system be responsive to CO.sub.2 liberated as a consequence of metabolism as well as to the inert reference gas, complicating the instrumentation required for such measurement. The concentration of the inert gas present in the culture gas used with such a system must be known and reproducible from lot to lot of culture gas, further complicating the overall detection system.
The use of radioisotopes or stable isotopes, or the use of inert reference materials has generally been considered necessary in order to provide for the detection of small quantities of gases produced by metabolism. Of the various gases produced by bacterial metabolism, carbon dioxide is the gas most commonly generated by the various families of bacteria, yeasts, and other primitive organisms. There thus exists a need for an instrumental system for measuring metabolically- produced carbon dioxide to detect bacteria and the like which does not require isotopic enrichment or labeling of nutrients and does not depend upon addition of any reference inert gas to the culture vial.
It is thus an object of the present invention to provide a rapid method for detecting the presence or absence of biologically active agents.
Another object of the invention is to provide a method of detecting the presence or absence of biologically active agents which uses comparatively inexpensive materials in conjunction with relatively straightforward instrumentation.
A further object of the invention is to provide an instrumental method for detecting the presence or absence of biologically active agents which is not subject to subjective interpretation.
An additional object of the present invention is to provide an instrumental system for detecting the presence or absence of biological activity which avoids the use of isotopically enriched or labeled nutrients, or the addition of inert material used as a reference for concentration ratio measurement.
It is yet a further object of the invention to provide an instrument system utilizing infrared analysis for the detection of biologically active agents which provides optimum detection sensitivity through matching of the head space carbon dioxide content of manufactured culture containers with the external culture gas supplied to the instrument for testing, calibration and purging purposes.